Methods of transforming a Listeria

ABSTRACT

Site-specific  Listeria  integration vectors and methods for their use are provided. The subject vectors include a bacteriophage integrase gene and a bacteriophage attachment site, where in many embodiments the bacteriophage that is the source of these elements is a listeriophage. In certain embodiments, the subject vectors further include a multiple cloning site, where the multiple cloning site may further include a polypeptide coding sequence, e.g., for a heterologous antigen. The subject vectors and methods find use in a variety of different applications, including the study of  Listeria  species and the preparation of  Listeria  vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/783,491, filed on May 19, 2010, now U.S. Pat. No. 8,192,991, which isa continuation of U.S. application Ser. No. 10/547,999, filed on Sep. 7,2006, now U.S. Pat. No. 7,749,510, which is a continuation-in-part ofPCT/US2003/013492, filed on Apr. 29, 2003, which is a continuation ofU.S. application Ser. No. 10/136,860, filed on Apr. 30, 2002, now U.S.Pat. No. 7,425,449; the disclosures of which are herein incorporated byreference.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos. 1 R37AI29619 and 1 R01 AI27655 awarded by the National Institute of Health.The Government has certain rights in this invention.

INTRODUCTION

1. Field of the Invention

The field of this invention is Listeria species, e.g., Listeriamonocytogenes, particularly recombinant strains of Listeria species, andmethods for their fabrication and use.

2. Background of the Invention

Listeria monocytogenes is a Gram-positive, food-borne human and animalpathogen responsible for serious infections in immunocompromisedindividuals and pregnant women. Severe L. monocytogenes infections inhumans are characterized by meningitis, meningoencephalitis, septicemia,and fetal death. L. monocytogenes is ubiquitous in nature and, inaddition, can be isolated from a wide variety of warm blooded animals.

Protocols for recombinantly engineering Listeria species are of interestin both research and therapeutic applications. For example, Listeriaspecies transformation protocols find use in the elucidation of themechanisms responsible for growth and virulence of these types ofbacteria. In addition, such protocols also find use in the preparationof live Listeria vaccines, which vaccines find use in a variety ofdifferent medical applications.

To date, a variety of different protocols have been employed totransform Listeria species, where such protocols include: homologousrecombination, transposon based recombination, etc. While differentprotocols are currently available for engineering Listeria species, suchmethods are not entirely satisfactory. Disadvantages currentlyexperienced with one or more of the available protocols include: (1)instability of the expression cassette in the transformed species; (2)variable impact on virulence of the transformed species; (3) sizeconstraints of the expression cassette that can be placed in thetransformed species; etc.

As such, despite the variety of different transformation protocolsavailable, there is continued interest in the identification of furthertransformation protocols that can expand the repertoire of availablegenetic tools. Of particular interest would be the development of anefficient, site-specific integration vector that was suitable for usewith a wide array of different Listeria species, where the vector didnot suffer from one or more of the above disadvantages of the currentlyavailable protocols.

RELEVANT LITERATURE

Patents and published patent applications of interest include: U.S. Pat.No. 5,830,702 and published PCT application serial nos.: WO 99/25376 andWO 00/09733.

SUMMARY OF THE INVENTION

Site-specific Listeria species integration vectors and methods for theiruse are provided. The subject vectors include a bacteriophage integrasegene and a bacteriophage attachment site, where in many embodiments thebacteriophage that is source of these elements is a listeriophage. Incertain embodiments, the subject vectors further include a multiplecloning site, where the multiple cloning site may further include acoding sequence, e.g., a coding sequence for a heterologous polypeptide,etc. The subject vectors and methods find use in a variety of differentapplications, including the study of Listeria species and thepreparation of Listeria species. vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A) Plasmid map of the pPL1 integration vector. SEQ ID NO:24provides the sequence of pPL1. Chloramphenicol resistance genes and E.coli origin of replication are shown in grey, RP4 origin of transfershown in white, integrase gene and L. monocytogenes p60 promoter shownin black. Multiple cloning site is shown at the bottom of the plasmidwith unique restriction sites noted below the MCS in a box. pPL24 andpPL25 inserts are shown schematically below the MCS and were cloned asdescribed in Materials and Methods. Final sizes of the plasmidconstructs and the restriction sites used in cloning are noted with eachof the inserts. (B) Plasmid map of the integration vector pPL2. SEQ IDNO:28 provides the sequence of pPL2. The color scheme and genes are thesame as in FIG. 1A except the PSA integrase and PSAattPP′ sites asnoted. The multiple cloning site with 13 unique restriction sites isshown at the bottom of the plasmid.

FIG. 2. Genomic organization of the attachment sites within the comKgene (A and B) and the tRNA^(Arg) gene (C and D). (A) Non-lysogenic L.monocytogenes strain, with an intact comK gene. Primers PL60 and PL61amplify across the bacterial attachment site comK-attBB′. (B) LysogenicL. monocytogenes strain (with approximately 40 kb of phage DNA insertedinto the comK gene) or integrated strain (with pPL1 construct insertedinto the comK gene). Primers PL14 and PL61 amplify across the hybridattachment site comK-attPB′. (C) L. monocytogenes serotype 4b strainnon-lysogenic at tRNA^(Arg)-attBB′ Primers NC16 and NC17 amplify acrossthe bacterial attachment site tRNA^(Arg)-attBB′ in serotype 4b strains.Asterisk indicates primers NC16 and NC17 are substituted with PL102 andPL103 to amplify across the bacterial attachment site tRNA^(Arg)-attBB′in serotype 1/2 strains. (D) Lysogenic L. monocytogenes strain (withapproximately 40 kb of phage DNA or 6 kb pPL2 vector DNA inserted at the3′ end of the tRNA^(Arg) gene. Primers NC16 and PL95 amplify across thehybrid attachment site tRNA^(Arg)-attBP′ in both serotype 4b and 1/2strains.

FIG. 3. Expression and functional complementation of ActA in SLCC-5764.(A) Coomassie blue stained SDS-PAGE of SLCC-5764 derived strains grownto late-log phase. ActA is indicated by an arrow. Lane 1: molecular sizemarker; lane 2: DP-L3780; lane 3: DP-L4083; lane 4: DP-L4086; lane 5:SLCC-5764; lane 6: DP-L4082; lane 7: DP-L4084; lane 8: DP-L4085; lane 9:DP-L4087. Strains are described in Table 1. (B) Actin tail formation andmovement of DP-L4087 in Xenopus cell extract. The top panel is a phaseimage; the bottom panel is a fluorescent image of the same field.

FIG. 4. (A) Clover-leaf diagram of tRNA^(Arg) utilized as the PSAattachment site. The arginine anticodon is circled. The region withsequence identity between the tRNA gene and the PSA attPP′ is outlined.The boundaries of the tRNA^(Arg) gene and Cove score (82.37) werepredicted with tRNAscan-SE (31). (B) Alignment of the tRNA^(Arg)-affBB′region of L. monocytogenes WSLC 1042 (top line) and the attPP′ region ofPSA downstream of the integrase gene. The 74 nt tRNA^(Arg) gene of L.monocytogenes is boxed and the 17 bp overlapping region of sequenceidentity (core integration site) is shaded grey. The tRNA^(Arg) geneanticodon is shown in bold and underlined. Identical nucleotide residuesare indicated by (:). The numbers located at the left indicate thenucleotide position in the DNA sequences of the WSLC 1042 attachmentsite (AJ314913) and PSA genome (AJ312240).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Site-specific Listeria species integration vectors and methods for theiruse are provided. The subject vectors include a bacteriophage integrasegene and a bacteriophage attachment site, where in many embodiments thebacteriophage that is source of these elements is a listeriophage. Incertain embodiments, the subject vectors further include a multiplecloning site, where the multiple cloning site may further include acoding sequence, e.g., for a heterologous polypeptide, etc. The subjectvectors and methods find use in a variety of different applications,including the study of Listeria species and the preparation of Listeriaspecies vaccines.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, representativemethods, devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing various invention components that aredescribed in the publications which might be used in connection with thepresently described invention.

In further describing the subject invention, the subject vectors arereviewed first in greater detail, followed by a review of the methods ofusing the subject vectors, as well as representative applications inwhich the subject vectors and methods find use.

Vectors

As summarized above, the subject invention provides Listeriasite-specific integration vectors, i.e., vectors that integrate intoListeria genomes in a site-specific manner. The subject vectors arecharacterized in that they stably integrate into a genome of a Listeriaspecies in a site-specific, as opposed to random, manner. As the subjectvectors integrate site-specifically into a target genome, the vectors ofthe subject invention typically are inserted into a specific location orsequence (i.e., domain, location) of the target genome by a mechanismmediated by a recombinase, specifically an integrase, where theintegrase is one that uses two distinct recognition sites as substrates,one of which is positioned in the integration site of the target genome(the site into which a nucleic acid is to be integrated) and anotheradjacent a nucleic acid of interest to be introduced into theintegration site (i.e., a site on the subject vector referred to hereinas the “bacteriophage attachment site”). For example, the recognitionsites for phage integrases are generically referred to as attB, which ispresent in the bacterial genome (into which nucleic acid is to beinserted) and attP (which is present in the phage nucleic acid adjacentthe nucleic acid for insertion into the bacterial genome, which isreferred to herein as the “bacteriophage attachment site”). Recognitionsites can be native (endogenous to a target genome) or altered relativeto a native sequence. Use of the term “recognize” in the context of anintegrase “recognizes” a recognition sequence, is meant to refer to theability of the integrase to interact with the recognition site andfacilitate site-specific recombination.

The subject vectors are capable of integrating into the genomes of awide variety of different Listeria species. Integration is readilydetermined by using any convenient assay, including those known to thoseof skill in the art, that can identify whether or not vector DNA hasintegrated into the genomic DNA of a target organism. For example,vector DNA can be introduced into a target organism, e.g., viaconjugation, and then integration can be determined via PCRamplification of the integrated sequence using primers flanking thetarget site, where the size of the amplification product isdeterminative of whether integration has occurred. An example of such anassay is provided in the experimental section, below. Additionalfeatures of many embodiments of the subject vectors is that theyreplicate autonomously in a non-Listeria host cell, e.g., E. coli, andare stable and innocuous in such non-Listeria host cells.

The subject integration vectors are typically double-stranded plasmidvectors, where the vectors are generally at least about 3 kb, often atleast about 5 kb and may be large as 15 kb, 20 kb, 25 kb, 30 kb orlarger, where the vectors sometimes range in size from about 3-6 toabout 20 kb. The vectors include a number of structural features thatimpart to the vectors the above-described functional characteristics.

One structural feature of the subject vectors is a bacteriophageintegrase gene, i.e., a nucleic acid coding sequence for a bacteriophageintegrase, which is operably linked to a Listeria specific promoter,such that the gene (coding sequence) is expressed in the Listeria cellfor which the vector is designed to be employed. In many embodiments,the bacteriophage integrase gene is one obtained from a listeriophage,i.e., it is a listeriophage integrase gene. A variety of differentlisteriophages are known in the art, where any convenient listeriophagemay serve as the source of the integrase gene, i.e., the integraseencoding nucleic acid. Specific integrases of interest include, but arenot limited to: the U153 integrase, the PSA integrase; and the like.

As indicated above, the integrase gene is operably linked to a promoter(as well as regulatory and/or signal sequences, if necessary) thatdrives expression of the integrase gene when the vector is present inthe Listeria cell for which the vector is designed and in whichintegration of the vector is desired. Any convenient promoter may beemployed, where in certain embodiments the promoter is a Listeriaspecific promoter. Representative promoters of interest include, but arenot limited to: the Listeria p60 promoter, the Listeria actA promoter,the Listeria plcA promoter, the Listeria mpl promoter, the Listeria plcBpromoter, the Listeria in/A promoter; a heat shock promoter; and thelike Promoters may also include certain bacteriophage promoters such asthe promoters for T7, Qβ, SP6 and the like if the strain of Listeriaalso expresses the cognate bacteriophage RNA polymerase. In addition tothe integrase gene/promoter element described above, the subject vectorsalso include a phage attachment site, i.e., a sequence or domain ofnucleotides that provide for a site-specific integration with a Listeriagenome. Any convenient phage attachment site may be employed, whereselection of the phage attachment site will depend, at least in part, onthe desired integration location for the vector. Representative phageattachment sites of interest include, but are not limited to: the comKintegration site (as described in greater detail in the experimentalsection below); the tRNA^(Arg) integration site (as described in greaterdetail in the experimental section below); and the like.

In addition, the subject vectors may include an origin of replicationthat provides for replication of the vector in a non-Listeria host cell,e.g., E. coli. This origin of replication may be any convenient originof replication or ori site, where a number of on sites are known in theart, where particular sites of interest include, but are not limited to:p15A; pSC101; ColEl; pUC; pMB9; and the like.

In addition, the subject vectors may include an origin of transfer siteor element when convenient, e.g., when the vector is introduced in thetarget Listeria cell using a conjugation protocol, as described ingreater detail below. Any convenient origin of transfer (oriT) may beemployed, where representative origins of transfer of interest include,but are not limited to: RP4 oriT; RSF1010 oriT;

and the like.

In addition, the subject vectors typically include at least onerestriction endonuclease recognized site, i.e., restriction site, whichis located on the vector at a location which is amenable to insertion ofa heterologous gene/expression cassette. A variety of restriction sitesare known in the art and may be present on the vector, where such sitesinclude those recognized by the following restriction enzymes: HindIII,PstI, SalI, AccI, HincII, XbaI, BamHI, SmaI, XmaI, KpnI, SacI, EcoRI,BstXI, EagI, NotI, SpeI and the like. In many embodiments, the vectorincludes a polylinker or multiple cloning site, i.e., a closely arrangedseries or array of sites recognized by a plurality of differentrestriction enzymes, such as those listed above.

In certain embodiments, the subject vectors include at least one codingsequence, e.g., a coding sequence for heterologous polypeptide/proteincoding sequence present in the multiple cloning site, e.g., as a resultof using a restriction endonuclease site present in the multiple cloningsite to insert the coding sequence into the vector, according to wellknown recombinant technology protocols. By “heterologous” is meant thatthe coding sequence encodes a product, e.g., a protein, peptide,polypeptide, glycoprotein, lipoprotein, or other macromolecule, that isnot normally expressed in Listeria. In many embodiments, this codingsequence is part of an expression cassette, which provides forexpression of the coding sequence in the Listeria cell for which thevector is designed. The term “expression cassette” as used herein refersto an expression module or expression construct made up of a recombinantDNA molecule containing at least one desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism, i.e., theListeria cell for which the vector is designed, such as thepromoter/regulatory/signal sequences identified above, where theexpression cassette may include coding sequences for two or moredifferent polypeptides, or multiple copies of the same coding sequence,as desired. The size of the coding sequence and/or expression cassettethat includes the same may vary, but typically falls within the range ofabout 25-30 to about 6000 bp, usually from about 50 to about 2000 bp. Assuch, the size of the encoded product may vary greatly, and a broadspectrum of different products may be encoded by the expressioncassettes present in the vectors of this embodiment.

The nature of the coding sequence and other elements of the expressioncassette may vary, depending on the particular application of thevector, e.g., to study Listeria species, to produce Listeria speciesvaccines, for cytosolic delivery of macromolecules, etc. For example,where the vectors are employed in the production of Listeria vaccines,the coding sequence may encode a heterologous antigen, whererepresentative heterologous antigens of interest include, but are notlimited to: (a) viral antigens, e.g., influenza np protein, HIV gagprotein, HIV env protein or parts thereof, e.g., gp120 and gp41, HIV nefprotein, HIV pol proteins, HIV reverse transcriptase, HIV protease,herpes virus proteins, etc., (b) malarial antigens; (c) fungal antigens;(d) bacterial antigens; (e) tumor and tumor related antigens; and thelike. Due to the flexibility of the vector system, virtually any codingsequence of interest may be inserted. Where secretion of the productencoded by the expression cassette is desired, the expression cassettemay include a coding sequence for a fusion protein of a selected foreignantigen and a protein that directs secretion, e.g., Listeriolysin O orPI-PLC; a signal sequence, such as hemolysin signal sequence, etc. Wherethe subject vectors are employed in the preparation of Listeria deliveryvehicles, e.g., as described in PCT publication no. WO 00/09733 (thepriority application of which is herein incorporated by reference); andDietrich et al., Nature Biotechnology (1998) 16: 181-185, theheterologous polypeptide coding sequence may be a cytolysin, e.g.,phospholipase, pore forming toxin, listeriolysin O, streptolysin O,perfringolysin O, acid activated cytolysins, phage lysins, etc. Othercoding sequences of interest include, but are not limited to: cytokines,costimulatory molecules, and the like. As indicated above, the vectormay include at least one coding sequence, where in certain embodimentsthe vectors include two or more coding sequences, where the codingsequences may encode products that act concurrently to provide a desiredresults.

In general, the coding sequence may encode any of a number of differentproducts and may be of a variety of different sizes, where the abovediscussion merely provides representative coding sequences of interest.

Methods of Preparing the Subject Vectors

The vectors of the subject invention may be produced using anyconvenient protocol, including by standard methods of restriction enzymecleavage, ligation and molecular cloning. One protocol for constructingthe subject vectors includes the following steps. First, purifiednucleic acid fragments containing desired component nucleotide sequencesas well as extraneous sequences are cleaved with restrictionendonucleases from initial sources. Fragments containing the desirednucleotide sequences are then separated from unwanted fragments ofdifferent size using conventional separation methods, e.g., by agarosegel electrophoresis. The desired fragments are excised from the gel andligated together in the appropriate configuration so that a circularnucleic acid or plasmid containing the desired sequences, e.g. sequencescorresponding to the various elements of the subject vectors, asdescribed above is produced. Where desired, the circular molecules soconstructed are then amplified in a host, e.g. E. coli. The proceduresof cleavage, plasmid construction, cell transformation and plasmidproduction involved in these steps are well known to one skilled in theart and the enzymes required for restriction and ligation are availablecommercially. (See, for example, R. Wu, Ed., Methods in Enzymology, Vol.68, Academic Press, N.Y. (1979); T. Maniatis, E. F. Fritsch and J.Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1982); Catalog 1982-83, NewEngland Biolabs, Inc.; Catalog 1982-83, Bethesda Research Laboratories,Inc. An example of how to construct a vector of the present invention isprovided in the Experimental section, below.

Methods

Also provided by the subject invention are methods of using the abovedescribed Listeria site specific integration vectors to integrate aheterologous nucleic acid into a Listeria genome. In practicing thesubject methods, a vector of the subject invention is introduced into atarget Listeria cell under conditions sufficient for integration of thevector into the target cell genome to occur. Any convenient protocol forintroducing the vector into the target cell may be employed.

Suitable protocols include: calcium phosphate mediated transfection;protoplast fusion, in which protoplasts harboring amplified amounts ofvector are fused with the target cell; electroporation, in which a briefhigh voltage electric pulse is applied to the target cell to render thecell membrane of the target cell permeable to the vector;microinjection, in which the vector is injected directly into the cell,as described in Capechhi et al, Cell (1980) 22: 479; and the like. Theabove in vitro protocols are well known in the art and are reviewed ingreater detail in Sambrook, Fritsch & Maniatis, Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press)(1989) pp16.30-16.55.

In certain embodiments, e.g., where direct introduction into the targetListeria cell does not provide optimal results, one representativemethod that may be employed is a conjugation method, which comprises:(a) introducing the vector into a non-Listeria host cell, e.g., E. coli;followed by (b) transfer of the vector from the non-Listeria host cellto the Listeria host cell, e.g., by conjugation. Introduction into thenon-Listeria host cell may be accomplished using any of the protocolsdescribed above. For the conjugation step, any convenient protocol maybe employed, where suitable protocols typically include combining thedonor and acceptor cells in a suitable medium and maintaining undersuitable conditions for conjugation and vector transfer to occur.Specific representative conditions are provided in the Experimentalsection below.

The above methods result in stable integration of the vector and anyexpression cassette carried thereby, e.g., that encodes a heterologousprotein/foreign antigen, into a target Listeria cell genome in a sitespecific manner. The subject methods find use with a wide variety ofdifferent Listeria species.

Utility

The above described vectors and methods of using the same find use in avariety of different applications, where representative applicationsinclude, but are not limited to: (a) research applications; (b) vaccinepreparation applications; (c) Listerial delivery vehicle preparationapplications; and the like.

One type of application in which the subject vectors and methods ofusing may be employed is in research of Listeria species. For example,the subject vectors and methods allow simple and efficient strainconstruction and are widely useful in various strains used to study theintracellular life cycle of L. monocytogenes. Additionally, the subjectvectors and methods may be employed to produce stable merodiploidstrains to allow refined copy number studies and studies of interactionswithin a protein through multimerization and testing of the dominance orrecessive nature of different alleles of a gene in the same bacterialstrain.

The subject vectors and methods also find use in vaccine preparationapplications. For example, the subject vectors find use in theproduction of Listeria cultures capable of expressing a heterologousantigen, i.e., Listeria vaccines, where the Listeria cells employed maybe attenuated. The attenuated strains employed may be capable of normalinvasion of a host cell, but incapable of normal survival or growth inthe cell or cell-to-cell spread, or they may have other alterations thatpreclude normal pathogenicity.

The vaccines produced using vectors of the present invention areadministered to a vertebrate by contacting the vertebrate with asublethal dose of the genetically engineered Listeria vaccine, wherecontact typically includes administering the vaccine to the host. Thusthe present invention provides for vaccines genetically engineered withthe integration vector and provided in a pharmaceutically acceptableformulation. Administration can be oral, parenteral, intranasal,intramuscular, intravascular, direct vaccination of lymph nodes,administration by catheter or any one or more of a variety of well-knownadministration routes. In farm animals, for example, the vaccine may beadministered orally by incorporation of the vaccine in feed or liquid(such as water). It may be supplied as a lyophilized powder, as a frozenformulation or as a component of a capsule, or any other convenient,pharmaceutically acceptable formulation that preserves the antigenicityof the vaccine. Any one of a number of well known pharmaceuticallyacceptable diluents or excipients may be employed in the vaccines of theinvention. Suitable diluents include, for example, sterile, distilledwater, saline, phosphate buffered solution, and the like. The amount ofthe diluent may vary widely, as those skilled in the art will recognize.Suitable excipients are also well known to those skilled in the art andmay be selected, for example, from A. Wade and P. J. Weller, eds.,Handbook of Pharmaceutical Excipients (1994) The Pharmaceutical Press:London. The dosage administered may be dependent upon the age, healthand weight of the patient, the type of patient, and the existence ofconcurrent treatment, if any. The vaccines can be employed in dosageforms such as capsules, liquid solutions, suspensions, or elixirs, fororal administration, or sterile liquid for formulations such assolutions or suspensions for parenteral, intranasal intramuscular, orintravascular use. In accordance with the invention, the vaccine may beemployed, in combination with a pharmaceutically acceptable diluent, asa vaccine composition, useful in immunizing a patient against infectionfrom a selected organism or virus or with respect to a tumor, etc.Immunizing a patient means providing the patient with at least somedegree of therapeutic or prophylactic immunity against selectedpathogens, cancerous cells, etc.

The subject vaccines prepared with the subject vectors find use inmethods for eliciting or boosting a helper T cell or a cytotoxic T-cellresponse to a selected agent, e.g., pathogenic organism, tumor, etc., ina vertebrate, where such methods include administering an effectiveamount of the Listeria vaccine. The subject vaccines prepared with thesubject vectors find use in methods for eliciting in a vertebrate aninnate immune response that augments the antigen-specific immuneresponse. Furthermore, the vaccines of the present invention may be usedfor treatment post-exposure or post diagnosis. In general, the use ofvaccines for post-exposure treatment would be recognized by one skilledin the art, for example, in the treatment of rabies and tetanus. Thesame vaccine of the present invention may be used, for example, both forimmunization and to boost immunity after exposure. Alternatively, adifferent vaccine of the present invention may be used for post-exposuretreatment, for example, such as one that is specific for antigensexpressed in later stages of exposure. As such, the subject vaccinesprepared with the subject vectors find use as both prophylactic andtherapeutic vaccines to induce immune responses that are specific forantigens that are relevant to various disease conditions.

The patient may be any human and non-human animal susceptible toinfection with the selected organism. The subject vaccines will findparticular use with vertebrates such as man, and with domestic animals.Domestic animals include domestic fowl, bovine, porcine, ovine, equine,caprine, Leporidate (such as rabbits), or other animal which may be heldin captivity.

In general, the subject vectors and methods find use in the productionof vaccines as described U.S. Pat. No. 5,830,702, the disclosure ofwhich is herein incorporated by reference; as well as PCT publication noWO 99/25376, the disclosures of the priority applications of which areherein incorporated by reference.

The subject vectors also find use in the production of listerialdelivery vehicles for delivery of macromolecules to target cells, e.g.,as described in: PCT publication no. WO 00/09733 (the priorityapplication of which is herein incorporated by reference); and Dietrichet al., Nature Biotechnology (1998) 16: 181-185. A variety of differenttypes of macromolecules may be delivered, including, but not limited to:nucleic acids, polypeptides/proteins, etc., as described in thesepublications.

Kits & Systems

Also provided are kits and systems that find use in preparing thesubject vectors and/or preparing recombinant Listeria cells using thesubject vectors and methods, as described above. For example, kits andsystems for producing the subject vectors may include one or more of: aninitial vector with a multiple cloning site; a restriction endonucleasefor cleaving a site in the multiple cloning site, a vector including anexpression cassette of interest which is to be inserted into themultiple cloning site; etc. Where the kits and systems are designed forthe production of the recombinant Listeria cells, the kits and systemsmay include vectors, or components for making the same, as describedabove, Listeria target cells, non-Listeria host cells, and the like. Thesubject kits may further include other components that find use in thesubject methods, e.g., reaction buffers, growth mediums, etc.

The various reagent components of the kits may be present in separatecontainers, or may all be precombined into a reagent mixture forcombination with template DNA.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL I. Materials and Methods

A. Construction of pPL1 Integration Vector

Standard molecular techniques were used in the construction of the 6101bp integration vector pPL1 (FIG. 1A). The complete sequence of the pPL1vector is provided as SEQ ID NO:25. pPL1 is a low copy plasmid thatreplicates autonomously in E. coli and integrates in a site-specificmanner in L. monocytogenes, and was assembled from 6 independent DNAsources as follows. Restriction sites in the PCR primers used forconstruction are underlined. All PCR reactions used in cloning stepsutilized Vent DNA polymerase (New England Biolabs).

The multiple cloning site (MCS) from pBluescript KS— (Alting-Mees, M.A., and J. M. Short. 1989. pBluescript II: gene mapping vectors. NucleicAcids Res. 17:9494.) (bp 1-171) was cloned after PCR with primers5′-GGACGTCATTAACCCTCACTAAAGG-3′ and 5′-GGACGTCAATACGACTCACTATAGG-3′ (SEQID NOS: 01 & 02). The low copy Gram-negative origin of replication andchloramphenicol acetyltransferase (CAT) gene from pACYC184 (Chang, A.C., and S. N. Cohen. 1978. Construction and characterization ofamplifiable multicopy DNA cloning vehicles derived from the P15A crypticminiplasmid. J. Bacteriol. 134:1141-1156.) (bp 172-2253) were clonedafter PCR with primers 5′-GGACGTCGCTATTTAACGACCCTGC-3′ and5′-GAGCTGCAGGAGAATTACAACTTATATCGTATGGGG-3′ (SEQ ID NOS: 03 & 04). Fordirect conjugation from E. coli to L. monocytogenes, the RP4 origin oftransfer (oriT) (Pansegrau, W., E. Lanka, P. T. Barth, D. H. Figurski,D. G. Guiney, D. Haas, D. R. Helinski, H. Schwab, V. A. Stanisich, andC. M. Thomas. 1994. Complete nucleotide sequence of Birmingham IncPalpha plasmids. Compilation and comparative analysis. J. Mol. Biol.239:623-663) (bp 2254-2624) was cloned from plasmid pCTC3 (Williams, D.R., D. I. Young, and M. Young. 1990. Conjugative plasmid transfer fromEscherichia coli to Clostridium acetobutylicum. J. Gen. Microbiol.136:819-826) after PCR with primers5′-GCACTGCAGCCGCTTGCCCTCATCTGTTACGCC-3′ and5′-CATGCATGCCTCTCGCCTGTCCCCTCAGTTCAG-3′ (SEQ ID NOS: 05 & 06). Thelisteriophage U153 integrase gene and attachment site (attPP′) (A.Nolte, P. Lauer, and R. Calendar, unpublished; by 2629-4127) (SEQ ID NO:25 includes the sequences for these sites), that direct thesite-specific integration of the plasmid were cloned after PCR withprimers 5′-GTAGATCTTAACTTTCCATGCGAGAGGAG-3′ and5′-GGGCATGCGATAAAAAGCAATCTATAGAAAAACAGG-3′ (SEQ ID NOS: 07 & -08). Forexpression of the U153 integrase gene, the L. monocytogenes p60 promoter(Lauer, P., J. A. Theriot, J. Skoble, M. D. Welch, and D. A. Portnoy.2001. Systematic mutational analysis of the amino-terminal domain of theListeria monocytogenes ActA protein reveals novel functions inactin-based motility. Mol. Microbiol. 42:1163-1177.) was PCR amplifiedwith primers 5′-CCTAAGCTTTCGATCATCATAATTCTGTC-3′ and5′-GGGCATGCAGATCTTTTTTTCAGAAAATCCCAGTACG-3′ (SEQ ID NOS: 09 & 10) andcloned upstream of the integrase gene. Base pairs 4570-6101 are a HindIII-Aat II restriction fragment subcloned from pUC18-Cat (a kind giftfrom Nancy Freitag), and contain the inducible Gram-positive CAT genefrom pC194 (Horinouchi, S., and B. Weisblum. 1982. Nucleotide sequenceand functional map of pC194, a plasmid that specifies induciblechloramphenicol resistance. J. Bacteriol. 150:815-825) (bp 4788-5850).

B. Cloning of the hly and actA Genes into pPL1.

The hly gene was subcloned from the plasmid pDP-906 (Jones, S., and D.A. Portnoy. 1994. Characterization of Listeria monocytogenespathogenesis in a strain expressing perfringolysin O in place oflisteriolysin O. Infect. Immun. 62(12):5608-5613.) by restrictiondigestion with BamH I and Xba I, gel purifying a 2.9 kb fragment and byligating it into pPL1 cut with BamH I and Spe I. The resultant plasmidwas designated pPL24 (FIG. 1A). The actA gene was PCR amplified from10403S genomic DNA with primers 5′-GGTCTAGATCAAGCACATACCTAG-3′ and5′-CGGGATCCTGAAGCTTGGGAAGCAG-3′(SEQ ID NOS:11 & 12). The 2220 bp PCRproduct was gel purified, cut with BamH I and Xba I, and cloned intopPL1 cut with BamH I and Spe I. The resultant plasmid was designatedpPL25 (FIG. 1A).

C. Phage Curing, Conjugation and Molecular Confirmation of PlasmidIntegration.

Phage curing was accomplished by adapting historical methodologies(Cohen, D. 1959. A variant of Phage P2 Originating in Escherichia coli,strain B. Virology 7:112-126; Six, E. 1960. Prophage substitution andcuring in lysogenic cells superinfected with hetero-immune phage. J.Bacteriol. 80:728-729.). L. monocytogenes 10403S derivatives carrying aprophage at comK-attBB′ (integrated in the comK ORF as described(Loessner, M. J., R. B. Inman, P. Lauer, and R. Calendar. 2000. Completenucleotide sequence, molecular analysis and genome structure ofbacteriophage A118 of Listeria monocytogenes: implications for phageevolution. Mol. Microbiol. 35(2):324-340.)) were grown in BHI at 37° C.to 10⁸ CFU/ml and infected with listeriophage U153 (Hodgson, D. A. 2000.Generalized transduction of serotype 1/2 and serotype 4b strains ofListeria monocytogenes. Mol. Microbiol. 35(2):312-323.) at amultiplicity of infection of 20:1 in the presence of 5 mM CaCl₂.Cultures were incubated by shaking at 37° C. for 75 minutes andinhibition of growth was monitored by comparison of the OD₆₀₀ of theinfected culture with an uninfected control culture. The infectedculture was diluted 10⁻² and 10⁻⁴ in BHI, and both dilutions were grownat 37° C. until the 10⁻² dilution had increased 100-fold in opticaldensity. The 10⁻⁴ fold dilution was then diluted 10⁻², and 3 μl wereplated on BHI. Fifty colonies were tested for phage release initially bytoothpicking colonies into 0.25 ml LB broth and replica plating at 30°C. on a lawn of Mack-4R (DP-L862), a non-lysogenic rough strain of L.monocytogenes particularly susceptible to forming plaques. Candidatesthat did not plaque were then tested by spotting 10 μl of culture on alawn of Mack-4R to detect plaque formation. If this second test wasnegative, the candidate was tested whether it could support plaqueformation by the phage from the parent 10403S strain (Φ10403, (Hodgson,D. A. 2000. Generalized transduction of serotype 1/2 and serotype 4bstrains of Listeria monocytogenes. Mol. Microbiol. 35(2):312-323.)).Curing was confirmed molecularly by PCR with the comK-attBB′ specificprimer pair PL60/PL61 (sequences follow) for the absence of a phage atcomK-attBB′. Approximately 10% of colonies were cured using thisprocedure.

Recipient strains of L. monocytogenes (SLCC-5764, DP-L1169 and DP-L1172)were made streptomycin resistant for counter-selection in conjugationexperiments by plate selection on BHI supplemented with 200 μg/mlantibiotic.

pPL1 plasmid constructs were electroporated into E. coli strain SM10(Simon, R., U. Priefer, and A. Pühler. 1983. A broad host rangemobilization system for in vitro genetic engineering: transposonmutagenesis in Gram negative bacteria. Bio/Technology 1:784-791.) usingstandard techniques (Sambrook, J., T. Maniatis, and E. F. Fritsch. 1989.Molecular Cloning: A Laboratory Manual, 2nd edn. ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). Bacterial strains weregrown to mid-log(OD₆₀₀˜0.55) shaking at 30° C. E. coli donor strainswere grown in LB containing 25 μg/ml of chloramphenicol, L.monocytogenes recipient strains were grown in BHI. 2.5 ml of donorculture was mixed with 1.5 ml of recipient culture and filtered ontopre-washed 47 mm 0.45 μm HA type filers (Millipore). The filter waswashed once with 10 ml BHI, transferred to a BHI plate with noantibiotics and incubated for 2 hours at 30° C. The bacterial cells weregently resuspended in 2.5 ml of BHI and 25 μl and 50 μl aliquots wereplated in 3 ml of LB top agar on BHI plates supplemented with 7.5 μg/mlchloramphenicol and 200 μg/ml streptomycin. Plates were incubated at 30°C. overnight and shifted to 37° C. for 2-3 days. Individual colonieswere picked and screened by PCR for integration at the phage attachmentsite using the primers PL14 (5′-CTCATGAACTAGAAAAATGTGG-3′) (SEQ IDNO:13), PL60 (5′-TGAAGTAAACCCGCACACGATG-3′) (SEQ ID NO:14) and PL61(5′-TGTAACATGGAGGTTCTGGCAATC-3′) (SEQ ID NO:15). PCR reactions wereperformed on small portions of individual bacterial colonies picked withsterile P200 pipet tips from BHI plates directly into 20 μl PCRreactions. The primer pair PL14/PL61 specifically amplifies attBP′ in aPCR reaction, resulting in a 743 bp product on integrated strains (bothprophage and pPL1 derivatives). The primer pair PL60/PL61 specificallyamplifies comK-attBB′ in a PCR reaction, resulting in a 417 bp productonly on non-lysogenic strains (i.e. DP-L4056). PCR assays were performedin a Hybaid Omn-E thermocycler with an annealing temperature of 55° C.for 30 cycles. Integrants arose at a frequency of approximately 2×10⁻⁴per donor cell.

D. Hemolysis on Blood Plates and Hemolytic Activity Assay.

Hemolysis on blood plates was scored on tryptic soy agar platessupplemented with 5% defimbrinated sheep blood (HemoStat, Davis Calif.).Hemolytic assays were performed essentially as described (Portnoy, D.A., P. S. Jacks, and D. J. Hinrichs. 1988. Role of hemolysin for theintracellular growth of Listeria monocytogenes. J. Exp. Med.167(4):1459-1471.). Hemolytic activity is expressed as the reciprocal ofthe dilution of culture supernatant required to lyse 50% of sheeperythrocytes.

E. Plaquing in L2 Cells.

Plaque sizes were determined as previously described (Lauer, P., J. A.Theriot, J. Skoble, M. D. Welch, and D. A. Portnoy. 2001. Systematicmutational analysis of the amino-terminal domain of the Listeriamonocytogenes ActA protein reveals novel functions in actin-basedmotility. Mol. Microbiol. 42(5):1163-1177.). Each strain was plagued in6 to 8 independent experiments and compared to 10403S in eachexperiment.

F. SDS-PAGE of Surface Expressed ActA.

Surface expressed ActA protein was prepared from late-log phasebacterial cultures grown in LB broth (OD₆₀₀˜0.7) by resuspendingequivalent amounts in SDS-PAGE buffer and boiling for 5 min. whichextracts surface-expressed proteins but does not perturb the cell wall.Equivalent amounts were loaded on 7% SDS-PAGE and visualized withCoomassie blue.

G. Xenopus laevis Cell Extract Motility Assays.

X. laevis egg cytoplasmic extract was prepared as described (Theriot, J.A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J.Mitchison. 1994. Involvement of profilin in the actin-based motility ofL. monocytogenes in cells and in cell-free extracts. Cell76(3):505-517.) and supplemented with tetramethylrhodamineiodoacetamide-labeled actin (Theriot, J. A., and D. C. Fung. 1998.Listeria monocytogenes-based assays for actin assembly factors. MethodsEnzymol. 298:114-122.). SLCC-5764-derived strains were grown overnightto stationary phase, washed 1×, added to cell extracts and incubated for45 minutes before microscopic observation.

H. LD₅₀ Determinations.

Limited LD₅₀ were performed in BALB/c mice as described (Portnoy, D. A.,P. S. Jacks, and D. J. Hinrichs. 1988. Role of hemolysin for theintracellular growth of Listeria monocytogenes. J. Exp. Med.167(4):1459-1471.). Animal experiments were performed in the laboratoryof Archie Bouwer at Oregon Health Sciences Center, Portland, Oreg.

I. Identification of the PSA Attachment Site and Construction of pPL2.

The PSA attachment site (tRNA^(Arg)-attBB′) DNA sequence was obtainedthrough a combination of inverse PCR and genome walking. Inverse PCR wasperformed on Sau3 AI-digested DP-L4061 DNA (WSLC 1042 lysogenic for PSA)(SEQ ID NO:26 is the sequence of 2,072 bp surrounding WSLC 1042tRNA^(Arg)-attBB′) using the divergent primers PL95(5′-ACATAATCAGTCCAAAGTAGATGC) (SEQ ID NO:16) and PL97(5′-ACGAATGTAAATATTGAGCGG) (SEQ ID NO:17) that anneal within the PSA intgene. The resultant DNA sequence was used to design furtheroligonucleotides and these were used with the Genome Walker kit(Clontech), per the manufacturer's recommendations. DNA sequence andtRNA analysis was done with using MacVector (Accelrys), DNAsis(Hitachi), BLAST algorithm (2), and tRNAscan-SE (Lowe, T. M., and S. R.Eddy. 1997. tRNAscan-SE: a program for improved detection of transferRNA genes in genomic sequence. Nucleic Acids Res. 25(5):955-964.).

pPL1 was modified to utilize a different attachment site on the L.monocytogenes chromosome by replacing the U153 integrase gene andattachment site in the plasmid. The PSA int and attPP′ were PCRamplified from PSA genomic DNA with primers PL100(5′-GAAGATCTCCAAAAATAAACAGGTGGTGG) (SEQ ID NO:18) and PL101(5′-CATGCATGCGTGGAGGGAAAGAAGAACGC) (SEQ ID NO:19) with Vent DNApolymerase, digested with Bgl II and Sph I, and ligated to pPL1 that hadbeen digested with the same enzymes. The resultant plasmid wasdesignated pPL2 (FIG. 1B). The sequence of pPL2 is provided as SEQ IDNO:28.

The DNA sequence of the PSA tRNA^(Arg)-affBB′ from serotype 1/2 L.monocytogenes strains was obtained by a plasmid-trap strategy. DP-L4211(pPL2 integrated in 10403S) genomic DNA was digested with Nsi I and NheI, which do not cleave in the vector, and ligated under diluteconditions to promote self-ligation. The ligations were transformed intoXL1-blue and chloramphenicol resistant colonies were selected. Theplasmids obtained were sequenced with the convergent primers PL94(5′-GGAGGGAAAGAAGAACGC) (SEQ ID NO:20) and PL95 (sequence above) forattPB′ and attBP′, respectively, which flank attPP′ in the PSA genomicDNA sequence. Further, because of the divergence between the sequencesdownstream of the tRNA^(Arg) gene among serotypes, a serotype 1/2specific PCR assay across tRNA^(Arg)-attBB′ was developed from the10403S DNA sequence and used to determine the prophage status of variousL. monocytogenes strains. Primers PL102 (5′-TATCAGACCTAACCCAAACCTTCC)(SEQ ID NO:21) and PL103 (5′-AATCGCAAAATAAAAATCTTCTCG) (SEQ ID NO:22)specifically amplify a 533 bp PCR product in non-lysogenic serotype 1/2strains. The primer pair NC16 (5′-GTCAAAACATACGCTCTTATC) (SEQ ID NO:23)and PL95 specifically amplify a 499 bp PCR product in strains that areeither lysogenic or contain an integration vector at tRNA^(Arg)-attBB′.(SEQ ID NO:27 provides the 643 bp surrounding 10403S tRNA^(Arg)-attBB′)

II. Results and Discussion

A. pPL1 Forms Stable, Single-Copy Integrants in Various L. monocytogenesStrains.

pPL1 is the first shuttle integration vector that we constructed tofacilitate strain construction in L. monocytogenes. In order to test thepPL1 vector, we needed a L. monocytogenes strain that did not have aphage at the comK bacterial attachment site. We adapted historicalmethods to cure L. monocytogenes strains of their prophages and foundafter superinfection with phage U153, which has the same attachment siteas the endogenous 10403S prophage, we were able to isolate prophage-freestrains (see Materials and Methods). The prophage-cured 10403S strainwas designated DP-L4056 and was used in subsequent experiments.

Conjugation was chosen as a method for introducing the vector into thetarget cells, as many Listeria spp. are inefficiently transformed.Conjugation of pPL1 from E. coli into L. monocytogenes was successful.Drug resistant transconjugants arose at a reproducible frequency of˜2×10⁻⁴ per donor E. coli cell, approximately three-fold lower thanconjugation with autonomously replicating plasmids, indicating ˜30%integration efficiency for strains receiving the plasmid by conjugation.All chloramphenicol resistant colonies were positive with the PCR assayusing primers PL14 and PL61 and negative using a PCR assay across attPP′in pPL1 (PL14 paired with a primer in the RP4 oriT) indicating that theywere true integrants and that the full genetic complement of plasmidpPL1 had integrated into the Listeria chromosome. In addition, thisexperiment demonstrated that pPL1 was not maintained as an episomalplasmid and that the vector did not integrate as a concatamer.

We tested the stability of the integrants under non-selective growthconditions. Three integrant strains, DP-L4074 and the merodiploidstrains DP-L4076 and DP-L4078 (described in the following sections) werepassed in liquid BHI media for 100 generations and plated for singlecolonies. Ninety-six colonies were then exposed to 0.1 μg/mlchloramphenicol (to induce CAT gene expression) and patched on platescontaining 7.5 μg/ml antibiotic. All colonies retained drug resistance.Thirty colonies from each non-selective growth experiment were assayedwith the PL14/PL61 PCR assay and all PCR reactions resulted in the 743bp product, indicating all transconjugants retained the integratedplasmid.

We further addressed whether the integration vector would be generallyuseful for any L. monocytogenes strain with an available attachmentsite. There have been greater than 320 listeriophages isolated, and manyhave restricted host ranges. It was unclear whether there was abiological barrier to U153 integrase gene function in host strains thatdo not support U153 infection. We therefore picked three strains thatdid not contain a prophage at the comK attachment site; two serotype 4bclinical isolates and SLCC-5764, a serotype 1/2a strain thatconstitutively expresses the known virulence factors in an unregulatedmanner and has been useful for studying these virulence factors invitro. Each of these strains was first made streptomycin resistant forcounter selection in conjugation experiments (as described in Materialsand Methods). Streptomycin resistant derivatives were chosen on thebasis of having the same growth rate as the parent strain to avoidexperimental complications related to viability. The resultant strains,DP-L4088, DP-L4089, and DP-L4082 all proved suitable recipients for pPL1integration at a similar frequency to DP-L4056.

Finally, we conducted a survey of L. monocytogenes isolates to identifysuitable strains that do not harbor a prophage at the comK attachmentsite using the PCR assays across comK-attBB′ (primers PL60/PL61) and thehybrid attPB′ (primers PL14/PL61). The results of these experiments(Table 2) indicated many of the strains commonly used to study thebiology and pathogenesis of L. monocytogenes including 10403S, L028, andEGDe had a prophage at comK.

TABLE 2 Prophage status of various strains PL60/PL61 PL14/PL61 StrainDescription Source serotype comK attPB' 10403S wild type rabbit pellets1/2a −^(a) + DP-L4056 10403S phage cured This work 1/2a +^(b) − DP-L861SLCC-5764 (Mack) WT (overexpresser) 1/2a + − DP-L3818 WSLC 1118::A118Camembert cheese 4b − + DP-L3633 EGDe WT (1960s human) 1/2a − + DP-L3293LO28 WT (clinical origin) 1/2c − + DP-L185 F2397 L.A., Jalisco cheese4b + − DP-L186 ScottA Massachusettes 4b + − outbreak, milk DP-L188 ATTC19113 Denmark, human 3 + − DP-L1168 clinical cole slaw 4b + − DP-L1169clinical patient 4b + − DP-L1170 clinical patient 4b + − DP-L1171clinical brie 1/2b + − DP-L1172 clinical alfalfa tablets 4b + − DP-L1173clinical deceased patient 4b − + DP-L1174 clinical deceased patient 4b− + DP-L3809 1981 Halifax placenta 4b + − DP-L3810 1981 Halifax CSF &brain 4b + − DP-L3812 1981 Halifax coleslaw 4b + − DP-L3813 1996 Halifaxblood ? − + DP-L3814 1981 Halifax CSF 4b + − DP-L3815 1993 Halifax CSF1/2a + − DP-L3816 1995 Halifax blood ? + − DP-L3817 1993 Halifax CSF1/2a + − DP-L3862 1998 Michigan patient 4b − + ^(a)(−): negative PCRresult for primer pair noted at top of column. ^(b)(+): positive PCRresult for primer pair noted at top of column. The PL60/PL61 primer pairspecifically amplify a 417 by PCR product in non-lysogenic strains andresult in no PCR product in lysogenic strains. The PL14/PL61 primer pairspecifically amplify a 743 by PCR product in lysogenic strains andresult in no PCR product in non-lysogenic strains.B. The Status of comK Did not Affect the Virulence of L. monocytogenes.

We next compared DP-L4056 and DP-L4074 to wild-type 10403S in standardvirulence assays to determine if the presence of a prophage at comK,lack of prophage, or integration vector altered the virulencephenotypes. These three strains were assayed for LLO activity, abilityto form plaques in monolayers of L2 cells, and for virulence in themouse LD₅₀ assay (Table 3). All were indistinguishable from one another,strongly suggesting that the integrity of the comK ORF and the presenceof pPL1 had no measurable impact on virulence.

TABLE 3 Complementation of actA and hly Hemolysis on blood HemolyticPlaque Strain Genotype plates activity^(a) size^(b) LD₅₀ ^(c) 10403Swild type + nd 100 (na) ~2 × 10⁴ DP-L4056 10403S phage cured + 97 101(1.4) <1 × 10⁵ DP-L4074 DP-L4056 comK::pPL1 + 98  99 (1.4) <1 × 10⁵DP-L4027 DP-L2161 phage cured, Δhly − 0  0 (0)   1 × 10⁸ DP-L4075DP-L4027 Δhly, comK::pPL24 + 99  97 (3.9) <1 × 10⁵ DP-L4076 DP-L4056comK::pPL24 + 198  96 (2) nd DP-L4029 DP-L3078 phage cured, ΔactA nd nd 0 (0)   2 × 10⁷ DP-L4077 DP-L4029 ΔactA, comK::pPL25 nd nd  86 (4) <1 ×10⁵ DP-L4078 DP-L4056 comK::pPL25 nd nd  72 (6.8) nd ^(a)Hemolytic unitsdata shown is from one representative experiment. nd: not determined.^(b)Plaque size is the average of 8 to 10 independent experiments andshown as a percent of wild type (defined as 100%). Standard deviationsare shown in parentheses. na: not applicable. ^(c)LD₅₀s of 10403S andΔhly (DP-L2161) were determined in (37), the LD₅₀ of the ΔactA strain(DP-L1942, a smaller deletion within the actA ORF that does not supportactin nucleation at the bacterial surface) was determined in (4).C. Full Complementation of hly at the Phage Attachment Site.

Listeriolysin-O (LLO), the gene product of hly, is a secretedpore-forming cytolysin that is responsible for escape from themembrane-bound vacuole when L. monocytogenes first enters a host cell.LLO is absolutely required for the intracellular life cycle of L.monocytogenes and virulence. LLO activity can be measured by hemolyticactivity on red blood cells. Hly mutants fail to form plaques inmonolayers of L2 cells and are 5 logs less virulent in the mouse LD₅₀assay.

We cloned the hly structural gene into pPL1 and conjugated this plasmidfrom E. coli to phage-cured wild type and Δhly L. monocytogenesderivatives, resulting in DP-L4076 (an hly merodiploid) and DP-L4075(hly only at the phage comK-att site). These strains were tested forhemolytic activity on blood plates, for the relative amount of hemolyticunits secreted, ability to form a plaque in a monolayer of L2 cells, andvirulence in the mouse LD₅₀ assay (Table 3). The quantitativecomplementation of hly in the deletion strain background and thedoubling of hemolytic units produced in the merodiploid strain indicatetwo things. First, gene expression is not de facto affected by ectopicexpression at the comK chromosomal position. Second, the hly promoter isself-contained. Additionally, a two-fold increase in the amount of LLOis not deleterious to the virulence and intracellular life cycle of L.monocytogenes, at least as measured by plaquing.

D. Complementation of actA at the Phage Attachment Site ApproachesWild-Type Expression.

ActA, a second major L. monocytogenes virulence factor, is responsiblefor commandeering host cell actin-cytoskeletal factors used forintracellular bacterial motility. ActA is also absolutely required forbacterial pathogenesis as mutants in actA are both unable to spread fromcell-to-cell and form a plaque in a cell monolayer and are 3 logs lessvirulent than wild type. Additionally, ActA expression appears to bemore complex than that of LLO: there are two promoters that drive actAexpression. One is immediately upstream of the actA ORF and the secondis in front of the mpl gene upstream of actA.

We constructed several strains to evaluate the complementation of actAat the phage attachment site. The first group included makingsecond-site complemented (DP-L4077) and merodiploid (DP-L4078) strainsin the 10403S background. These were assayed for plaque formation in anL2 monolayer (Table 3). Integrated ActA did not fully complement in thisassay (plaque size of 86%) and the merodiploid strain formed an evensmaller plaque (72% of wild type). We interpret these results toindicate that there may be a small contribution of the second promoterupstream of the mpl gene for optimal actA expression. Additionally,there appears to be a critical concentration of ActA on the surface ofintracellular bacteria because two copies of actA (with 3 promotersdriving expression) further decreases the ability to spread from cell tocell, presumably because there is too much ActA on the bacterial surfacefor optimal motility.

We further tested ActA complementation in the virulence geneover-expressing strain SLCC-5764. ActA is effectively expressed in thisstrain from the comK-attBB′ site (FIG. 3A, lane 9). Considering theplaquing data of 10403S complemented actA strains, it may have beenpredicted that the merodiploid strain DP-L4085 would make more ActA thanthe parent strain. However, this was not observed: the parent strain,the complemented strain and the merodiploid strain all expressed similarlevels of ActA (FIG. 3A, lanes 5, 8, and 9). This observation was likelydue to the complete lack of regulation and high level of constitutiveexpression of ActA in SLCC-5764. Additionally, DP-L4087 supports actinnucleation at the bacterial surface, actin tail formation and bacterialmotility in cell extracts (FIG. 3B). The results of these cell-extractexperiments indicate that the integration vector system forcomplementation will be useful for in vitro studies of L. monocytogenesmotility, facilitating strain construction and placing various molecularconstructs in different host strains for study in a desired set ofassays. In particular, several alleles of actA that have unusualmotility phenotypes have been transferred to the SLCC-5764 ΔactA strainusing pPL1 and are currently being evaluated in cell extracts. The studyof these mutants in the simplified cell-extract system should yieldinsights into the activities of poorly understood regions of the ActAprotein.

The strains referenced above are provided in the following

Strain Relevant genotype or plasmid E. coli: SM10 Conjugation donor.F-thi-1 thr-1 leuB6 recA tonA21 lacY1 supE44 Mu + Cl-[RP4-2(Tc::Mu)]Km^(r) Tra+ XL1-Blue Plasmid manipulations. recA1 endA1 gyrA96 thi-1hsdR17 supE44 relA1 lac [F’ proAB lacI^(q)Z ΔM15 Tn10 (Tet^(r))]DP-E4067 Integration vector pPL1/SM10 DP-E4068 hly integration vectorpPL24/SM10 DP-E4069 actA integration vector pPL25/SM10 DP-E4190Integration vector pPL2/SM10 L. monocytogenes: 10403S wild type DP-L405610403S phage cured DP-L4027 DP-L2161 phage cured, Δhly DP-L4029 DP-L3078phage cured, ΔactA DP-L4074 DP-L4056 comK::pPL1 DP-L4075 DP-L4027 Δhly,comK::pPL24 DP-L4076 DP-L4056 comK::pPL24 DP-L4077 DP-L4029 ΔactA,comK::pPL25 DP-L4078 DP-L4056 comK::pPL25 SLCC-5764 Virulence gene overexpresser (Mack, DP-L861) DP-L862 Mack-4R (SLCC-5764 rough isolate)DP-L4082 SLCC-5764 Str^(r) derivative DP-L3780 SLCC-5764 ΔactA (deletionof amino acids 7-633) DP-L4083 DP-L3780S (Str^(r) dervivative) DP-L4084DP-L4082, comK::pPL1 DP-L4085 DP-L4082, comK::pPL25 DP-L4086 DP-L4083,comK::pPL1 DP-L4087 DP-L4083 ΔactA, comK::actA DP-L4088 DP-L1169S 4bstrain, Str^(r) DP-L4089 DP-L1172S 4b strain, Str^(r) DP-L4090 DP-L4088,comK::pPL1 DP-L4091 DP-L4089, comK::pPL1 DP-L4199 EGDe, Str^(r)derivative DP-L4026 WSLC 1042, (ATCC 23074) DP-L4061 WSLC 1042::PSADP-L4221 10403S, tRNA^(Arg)::pPL2E. Phage PSA Integrates into a tRNA^(Arg) Gene and pPL2 Construction.

pPL1 integration into L. monocytogenes strains that harbour a prophagein the comK attachment site is hindered by the process of first havingto cure the prophage from the host strain. To alleviate the need for thephage-curing step, the specificity of pPL1 integration was changed tothat of the PSA prophage. PSA, (Phage from ScottA) is the prophage of L.monocytogenes strain ScottA, a serotype 4b strain that was isolatedduring an epidemic of human listeriosis. Using the PSA genomic DNAsequence, we identified an integrase-like ORF with a contiguousnon-coding sequence that we predicted to contain the attPP′ sequences.The PSA integrase sequence was then used to obtain the DNA sequence ofPSA-attBB′ from the PSA lysogenic strain DP-L4061 (see Materials andMethods). PSA was found to integrate in a tRNA^(Arg) gene that is 88%identical to a tRNA^(Arg) gene (tmSL-ARG2) from B. subtilis. Theanticodon of the tRNA^(Arg) gene is 5′UCU, the most commonly usedarginine anticodon in L. monocytogenes. The PSA and bacterial attachmentsites share 17 bp of DNA identity, and the tRNA^(Arg)-attBB′ contains ashort nucleotide sequence that completes the tRNA^(Arg) sequence that isinterrupted by integration of PSA (FIG. 4). The attachment sitetRNA^(Arg) gene is present only once in the genome of L. monocytogenesstrain EGDe and apparently only once in the serotype 4b. This indicatesthat not only is the PSA integration site unique, but also that precisereconstitution of the gene upon integration (or excision) is likelyrequired for survival of the cell.

pPL2 was constructed by replacing the U153 listeriophage integrase geneand attachment site in pPL1 with the PSA listeriophage integrase geneand attachment site. pPL2 was transformed into SM10 and the resultingstrain was mated into 10403S, EGDe (carrying a streptomycin resistancemutation) and the serotype 4b strain DP-L4088. Chloramphenicol resistanttransconjugants arose from each of these crosses at approximately 2×10⁻⁴per donor cell, the same rate as pPL1 integration. Two recombinants fromeach cross were restreaked under drug selection and tested by PCR forthe presence of PSA-attBP′ using primers NC16 and PL95. The expected 499bp PCR product was obtained in each of the colonies tested, indicatingpPL2 integrates into tRNA^(Arg)-attBB′ in both serotype 1/2 and 4bstrains. We tested the stability of the integrated pPL2 in both EGDe andDP-L4088 strains with the same non-selective 100-generation experimentdescribed for pPL1. Forty-nine colonies from each of the amplifiedcultures were tested for chloramphenicol resistance. The EGDe-derivedstrains retained 100% drug resistant colonies indicating completestability of the integrants. In the case of the DP-L4088 integrant, twoof the 49 colonies were chloramphenicol sensitive, suggesting a lowlevel of excision can occur in this serotype 4b strain. In order to testwhether precise excision had occurred, we PCR amplified acrosstRNA^(Arg)-attBB′ and sequenced the PCR products. The wild-type DNAsequence was obtained, indicating a precise excision event.

During the course of our PCR experiments, we noted a divergence betweenthe tRNA^(Arg)-attPB′ sites from serotype 4b and serotype 1/2 L.monocytogenes. To determine nature of this divergence, we isolated andsequenced the tRNA^(Arg)-attBB′ site from 10403S (as described inMaterials and Methods). We found that the sequence of attPB′ in 10403S(3′ of the tRNA^(Arg) gene) is unrelated to that of the serotype 4bstrain WSLC 1042. In contrast to this, the sequence of attBP′ (5′ of thetRNA^(Arg) gene) in 10403S is 96-97% identical to the correspondingregions in L. monocytogenes serotype 4b strain WSLC 1042, the serotype4b strain sequenced by TIGR, and the serotype 1/2a strain EGDe sequencedby the European Listeria Consortium. Thus, the bacterial attBB′sequences recognized by the PSA integrase are likely to encompass moreof the attB DNA sequence than the attB′ DNA sequence. Additionally, wetested the availability of the tRNA^(Arg)-attBB′ in the commonlaboratory strains of L. monocytogenes with a PCR assay using primersPL102 and PL103. We found the tRNA^(Arg) attachment site to be availablein strains 10403S, EGEe, and L028 indicating that pPL2 may be readilyutilized in these backgrounds for strain construction, complementation,and genetics studies without first curing endogenous prophages.

F. Expression of Aquoria victoria GFP.

A GFP coding sequence as described in U.S. Pat. No. 5,777,079 (thedisclosure of which is herein incorporated by reference), is cloned intoplasmid pPL1, transferred into the genome of L. monocytogenes. The GFPcoding sequence is amplified by polymerase chain reaction (PCR) and thePCR fragment is cloned into the multiple cloning site of pPL1. Asuitable promoter, containing appropriate transcriptional elements and atranslational leader sequence for expressing the GFP in L. monocytogenesare cloned at the 5′ end of the GFP coding sequence such that theyinduce the expression of the GFP protein. The modified pPL1 plasmidconstructs are electroporated into E. coli strain SM10 using standardtechniques, and the modified pPL1 plasmid construct is conjugated intoL. monocytogenes as described above. Recombinant L. monocytogenes areselected on BHI plates supplemented with 7.5 μg/ml chloramphenicol and200 μg/ml streptomycin. Individual colonies are picked and screened byPCR for integration at the phage attachment site using the primers PL14(5′-CTCATGAACTAGAAAAATGTGG-3′) (SEQ ID NO:13), PL60(5′-TGAAGTAAACCCGCACACGATG-3′) (SEQ ID NO:14) and PL61(5′-TGTAACATGGAGGTTCTGGCAATC-3′) (SEQ ID NO:15). Cultures of recombinantL. monocytogenes are grown, prepared and screened for GFP.

III. Utility of pPL1 and pPL2

The construction and characterization of the first single step sitespecific integration vectors for use in L. monocytogenes furthers thegenetic tools available for the study of this pathogen. These vectorsallow more facile strain construction than historic methods and arewidely useful in various strains used to study the intracellular lifecycle of L. monocytogenes. Additionally, stable merodiploid strains canbe constructed to allow refined copy number studies and studies ofinteractions within a protein through multimerization and testing of thedominance or recessive nature of different alleles of a gene in the samebacterial strain.

pPL1 and pPL2 are also useful for vaccine development, e.g., for atleast enhancing, including both eliciting and boosting, an immuneresponse to a target cells or cells, e.g., a foreign pathogen, etc.Several recombinant L. monocytogenes systems have been used to elicitcell-mediated immune responses in mice (Frankel, F. R., S. Hegde, J.Lieberman, and Y. Paterson. 1995. Induction of cell-mediated immuneresponses to human immunodeficiency virus type 1 Gag protein by usingListeria monocytogenes as a live vaccine vector. J. Immunol.155(10):4775-4782.; Goossens, P. L., G. Milon, P. Cossart, and M. F.Saron. 1995. Attenuated Listeria monocytogenes as a live vector forinduction of CD8+ T cells in vivo: a study with the nucleoprotein of thelymphocytic choriomeningitis virus. Int. Immunol. 7(5):797-805;Ikonomidis, G., Y. Paterson, F. J. Kos, and D. A. Portnoy. 1994.Delivery of a viral antigen to the class I processing and presentationpathway by Listeria monocytogenes. J. Exp. Med. 180(6):2209-2218.; Shen,H., M. K. Slifka, M. Matloubian, E. R. Jensen, R. Ahmed, and J. F.Miller. 1995. Recombinant Listeria monocytogenes as a live vaccinevehicle for the induction of protective anti-viral cell-mediatedimmunity. Proc. Natl. Acad. Sci. USA 92(9):3987-3991.). One limitationwith plasmid-based expression of recombinant proteins in L.monocytogenes is the stability of the plasmids in vivo (i.e. in the hostanimal) without selection. Additionally, chromosomal construction ofstrains expressing foreign antigens is time consuming. pPL1 and pPL2alleviate both of these concerns.

It is evident from the above results and discussion that subjectinvention provides a number of advantages. The construction andcharacterization of the first single step site specific integrationvectors for use in L. monocytogenes as described herein furthers thegenetic tools available for the study of this pathogen. For example, thesubject vectors and methods allow more facile strain construction thanhistoric methods and are widely useful in various strains used to studythe intracellular life cycle of L. monocytogenes. Furthermore, thesubject invention provides important new tools for the production ofvaccine preparations. One limitation with plasmid-based expression ofrecombinant proteins in L. monocytogenes is the stability of theplasmids in vivo (i.e. in the host animal) without selection.Additionally, chromosomal construction of strains expressing foreignantigens is time consuming. The subject vectors and methods of usealleviate both of these concerns. As such, the present inventionrepresents a significant contribution to the art.

All publications and patent application cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A method of stably transforming a Listeria, saidmethod comprising: contacting said Listeria with an integration plasmidcomprising a listeriophage attachment site (attPP′) that is capable ofintegrase-mediated stable site-specific Listeria genome integrationunder conditions sufficient for said integration plasmid to integrateinto said Listeria's genome; wherein said conditions comprise alisteriophage integrase that mediates the integration of saidintegration plasmid into said genome; and wherein said genome comprisesa listeriophage integrase gene following said integration.
 2. The methodaccording to claim 1, wherein said integration plasmid comprises saidlisteriophage integrase gene.
 3. The method according to claim 1,wherein said attachment site provides for integration at an integrationsite selected from the group consisting of: the comK integration siteand the tRNA^(Arg) integration site.
 4. The method according to claim 1,wherein said integration plasmid further includes a multiple cloningsite.
 5. The method according to claim 4, wherein said integrationplasmid further includes a heterologous coding sequence.
 6. The methodaccording to claim 5, wherein said heterologous coding sequence encodesa polypeptide.
 7. The method according to claim 6, wherein saidpolypeptide is an antigen.
 8. The method according to claim 1, whereinsaid integration plasmid is pPL1.
 9. The method according to claim 1,wherein said integration plasmid is pPL2.
 10. The method according toclaim 1, wherein the listeriophage attachment site (attPP′) is selectedfrom the group consisting of U153 attPP′ and PSA attPP″.
 11. The methodaccording to claim 1, wherein: a) the listeriophage attachment site isU153 attPP′ and the integrase is U153 integrase, or b) the listeriophageattachment site is PSA attPP′ and the integrase is PSA integrase. 12.The method of claim 1, wherein said Listeria integrase gene is presentin an integrated prophage.
 13. The method of claim 12, wherein saidprophage is integrated at the comK gene.
 14. A method of stablytransforming a Listeria with a heterologous coding sequence, said methodcomprising: contacting said Listeria with a listeriophage integrase geneand an integration plasmid comprising a listeriophage attachment siteand the heterologous coding sequence under conditions sufficient forsaid integration plasmid to integrate into said Listeria's genome;wherein said genome comprises a listeriophage integrase gene followingsaid integration.
 15. The method according to claim 14, wherein thelisteriophage attachment site is the U153 attachment site (attPP′) orthe PSA attachment site (attPP′).
 16. The method according to claim 14,wherein said heterologous coding sequence encodes a polypeptide.
 17. Themethod according to claim 14, wherein said polypeptide is an antigen.18. A method of stably transforming a Listeria with a heterologouscoding sequence, said method comprising: contacting a Listeriacomprising a listeriophage integrase gene with an integration plasmidcomprising a listeriophage attachment site and the heterologous codingsequence.
 19. The method according to claim 18, wherein thelisteriophage attachment site is the U153 attachment site (attPP′) orthe PSA attachment site (attPP′).
 20. The method according to claim 18,wherein said heterologous coding sequence encodes a polypeptide.
 21. Themethod according to claim 18, wherein said polypeptide is an antigen.22. The method of claim 18, wherein said listeriophage integrase gene isintegrated into the genome of said Listeria.
 23. The method of claim 22,wherein said listeriophage integrase gene is present in an integratedprophage.
 24. The method of claim 23, wherein said prophage isintegrated at the comK gene.